Liposome-lentivirus for miRNA therapy with molecular mechanism study

Background Cancer stem cells (CSCs) play a vital role in the occurrence, maintenance, and recurrence of solid tumors. Although, miR-145-5p can inhibit CSCs survival, poor understanding of the underlying mechanisms hamperes further therapeutic optimization for patients. Lentivirus with remarkable transduction efficiency is the most commonly used RNA carrier in research, but has shown limited tumor-targeting capability. Methods We have applied liposome to decorate lentivirus surface thereby yielding liposome-lentivirus hybrid-based carriers, termed miR-145-5p-lentivirus nanoliposome (MRL145), and systematically analyzed their potential therapeutic effects on liver CSCs (LCSCs). Results MRL145 exhibited high delivery efficiency and potent anti-tumor efficacy under in vitro and in vivo. Mechanistically, the overexpressed miR-145-5p can significantly suppress the self-renewal, migration, and invasion abilities of LCSCs by targeting Collagen Type IV Alpha 3 Chain (COL4A3). Importantly, COL4A3 can promote phosphorylating GSK-3β at ser 9 (p-GSK-3β S9) to inactivate GSK3β, and facilitate translocation of β-catenin into the nucleus to activate the Wnt/β-catenin pathway, thereby promoting self-renewal, migration, and invasion of LCSCs. Interestingly, COL4A3 could attenuate the cellular autophagy through modulating GSK3β/Gli3/VMP1 axis to promote self-renewal, migration, and invasion of LCSCs. Conclusions These findings provide new insights in mode of action of miR-145-5p in LCSCs therapy and indicates that liposome-virus hybrid carriers hold great promise in miRNA delivery. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-024-02534-0.


Introduction
So far, several RNA-based therapies have shown substantial promise in cancer therapy and are under systematic evaluation for clinical translation.The RNAs used in these RNA therapies include antisense oligonucleotides (ASOs) [1], small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) [2], ASO antimiRs, miRNA mimics, miRNA sponges [3], therapeutic circular RNAs (circRNAs) [4] and CRISPR-Cas9-based gene editing [5].However, only few selected RNAs have been licensed for clinical usage and thus the complete potential of RNA therapies has not been harnessed so far.The main reason is that RNAs have certain intrinsically physical shortcomings, such as instability, negative charge and hydrophilic nature which can effectively inhibit their diffusion through the cell membranes [6,7].
Thus, to circumvent these difficulties and achieve efficient RNA delivery, both viral and non-viral delivery methods have been investigated till now.For the non-viral delivery, nanoparticle (NP)-mediated delivery has been extensively investigated because synthetic NP carriers have low immunogenicity, biocompatibility, and can be easily subjected to physical and chemical modifications to achieve optimal pharmacokinetics and pharmacodynamics [8].Although a great number of NP carriers including liposomes [9], peptides [10], polymers [11] inorganic NPs [12], and hydrogel [13,14] have been intensively reported in literature, so far only liposomes have successfully been translated in clinic with FDA approval for siRNA therapeutics (Table 1).It is primarily because of various outstanding features for liposomes, such as those of colloidal stability, ease in tuning composition and surficial properties, long term circulation, efficient internalization, good accumulation as well as penetration, and controlled release in tumors [15].As such, liposomes at present are being increasingly examined in clinical trails for miRNA and other RNA therapies.
Interestingly, in comparison to the non-viral methods, viruses are highly-evolved, natural delivery agents used as the genetic materials [17].In addition to their remarkable transduction efficiency, lentivirus vectors (LVs) generally display not only durable RNA expression RNA capability, but also possess low immunogenicity, which is appealing for RNA delivery.These factors have contributed to emergence of lentiviruses as a powerful tool for the studies of gene functions, which have been demonstrated in several previous publications [18][19][20].However, it also should be noted that generally lentivirus possess poor tumor cell-targeting capability because of their non-specific nature of transgene integration by viral integration machinery [21], which has markedly hampered optimal their optimal application for miRNA therapy.It is envisioned that the nanocarrier which can effectively integrate the merits of both liposome-based non-viral carriers and lentivirus could greatly boost the therapeutic efficacy of RNA, although the related carriers have not been reported so far.
Notably, among different RNAs used in tumor therapeutics, microRNAs (MiRNAs) with substantial capability in silencing RNA and regulating gene expression after transcription [22], has emerged as the rising star and gained increasing attention in the field of tumor therapeutics.However, the potential mechanisms of action for several miRNAs therapies are not well understood.Generally, miRNAs can target multiple genes within one specific pathway, and thus could trigger a broader yet specific response [23].Thus, to further improve the treatment outcomes for cancer patients, it could be of great help to not only study about the functioning mechanisms of miRNAs in tumor development, progression, and response to therapy could be useful [24], but also to formulate efficient carriers for miRNA delivery, which could significantly boost the development of advanced RNA-based therapeutic paradigms.
Eradication of cancer stem cells (CSCs) can greatly contribute to effective treatment of bulk cancer cells, since CSCs play an important role in initiation as well as progression, development of multiple drug resistance, metastasis, and recurrence of tumors [25].For instance, in hepatocellular carcinoma (HCC), successful ablation of liver CSCs (LCSCs) remains the key to treat HCC bearing patients and improve the prognosis [26].LCSCs are a subset of cells existing in liver cancer tissues with stem cell characteristics that can express CD133, CD90, CD44, and Ep-CAM [27].It has been previously reported that in HCC, miR-145-5p expression was significantly down-regulated and closely related to prognosis functioned as a putative oncosuppressor [28].Thus, the examination of specific roles and mechanisms of miR-145-5p in HCC could be an area of great interest.Moreover, the previous study has revealed that miR-145-5p can regulate the tumorigenicity of LCSCs and hamper the acquisition of pancreatic CSCs characteristics [29].However, the molecular mechanisms through which miR-145-5p can affect the tumorigenicity of LCSCs are still unknown, and thus it is important to thoroughly understand the underlying causes which can lead to novel therapeutic targets for miR-145-5p to better clinical outcomes.
It has also been established that autophagy in HCC is crucial for tumorigenesis, metastasis, targeted therapy, and drug resistance.Furthermore, autophagy can strengthen capacity of CSCs for self-renewal and extend their survival ability [30].In addition, the Wnt pathway activation can mediate tumor aggressiveness and promote EMT as well as metastasis of liver CSCs [31].In this study, we have not only synthesized miR-145-5pcontaining liposome-lentivirus hybrid carriers for efficient delivery of miR-145-5p, termed miR-145-5p-lentivirus nanoliposome (MRL145), but also systematically investigated the molecular role of miR-145-5p in both HCC development and LCSC functioning (Scheme 1).Liposomes were used to coat miR-145-5p loaded lentivirus, thus yielding the liposome-lentivirus hybrid carriers.Since HCCLM3 cells are enriched with stem-like cancer cells [32] and have higher metastatic potential than other liver cancer cell lines [33], so we have isolated liver cancer stem cells (LCSCs) from HCCLM3 cells using the microsphere culture method [34] for both in vitro and in vivo experiments.Furthermore, the therapeutic efficacy of MRL145 in eradicating LCSCs was also evaluated.The evidence suggests that autophagy is proposed to maintain stemness phenotype in cancer [35].Notably the therapeutic mechanisms of miR-145-5p were systematically

MiR-145-5p inhibited the biological behavior of LCSCs
To optimize RNA therapy, following key factors should be carefully considered \.(i) It should be extensively tested for immunogenicity.(ii) The carrier should be chemically modified to improve the pharmacokinetics as well as pharmacodynamics, and efficiently delivered to the lesion sites after consideration of biodistribution patterns and intracellular escape mechanisms.(iii) The RNA drug finally should be able to specifically and potently interact with the intended target and used at an optimal dose to trigger the desired therapeutic effect.It has been reported that electrostatic charge coupling of miRNAs to the cationic lipid of liposomes can facilitate the cellular uptake [36].Moreover, the capsid proteins of lentivirus are rich in basic amino acids, and the capsid protein molecule is mainly positively charged [37,38].While, the oybean lecithin is negatively charged [39].The positive-negative alternate adsorption effect was supposed to assist in the self-assembly of liposome, and consequently formed nanoparticles.For lentivirus, they are hydrophilic and could stay in the aqueous cores of liposomes.
To achieve efficient delivery of miR-145-5p under both in vitro and in vivo settings, we have formulated liposome-lentivirus hybrid carriers (MRL145), which could exhibit combinatorial merits of both liposome and lentivirus with significantly improved therapeutic efficacy.Briefly, over-expressed miR-145-5p lentivirus was encapsulated in a liposomal nanoparticle with a diameter of around 110 nm.The blank-liposome was then synthesized using a thin-film hydration method and ultrasonic treatment.Furthermore, the blank-liposome dispersion and miR-145-5p solution were mixed and extruded through a 220 nm polycarbonate membrane for 10 times, thereby yielding MRL145.Thereafter, we have examined the mean size distribution, surface charge, and vesicle morphology of MRL145 formulations.Transmission Electron Microscope (TEM) images (Fig. 1a) showed that the MRL145 nanoparticles possessed typically spherical and core-shell structure with an average particle size of 100 ± 2 nm, which was quite similar to that of the blank liposomes and opposite to lentivirus (Figure S1a, b).The surface charge of blank-liposome changed from 26.5 mV to 0.41 mV after encapsulation of miR-145-5p lentivirus (Figure S1c), whereas polydispersity index (PDI) for blank liposome and MRL145 was 0.218 and 0.257 (Figure S1d), respectively.These results indicated the successful coating of blank-liposome cloak on the surface of the miR-145-5p-loaded lentivirus.
As liposome has some passive targeting capability in cancer therapy [40,41].To validate whether the Scheme 1 Illustration of the synthesis route of MRL145 and mode of action of 145-5p therapeutics against HCC development and LCSCs functioning integration of liposome and lentivirus could be useful for MiRNA delivery, real-time fluorescence imaging was conducted to examine the potential targeting tumor capability of MRL145 in vitro (Fig. 1b) and in vivo (Fig. 1d), respectively.Firstly, we transfected HEK-293T cells, which represent tool cells for lentiviral packaging production, titer determination and cell transfection, with pGCMV/EGFP/miR-145 and liposome (miR-145), or miR-145-5p lentivirus or MRL145, respectively.After 48 h, it was observed that the transfection efficiency of the MRL145 group was significantly higher than that of the miR-145-5p lentivirus and the miR-145 group (Fig. 1b).Furthermore, the mRNA expression of miR-145-5p was significantly up-regulated in the MRL145 group in comparison to both the miR-145-5p lentivirus and the miR-145 group (Figure S1e).In vivo, a significant signal intensity was found in tumor at 9 h (Fig. 1c), thus revealing the maximal accumulation of MRL145.Furthermore, the mice were euthanized after injection within 24 h, and the HE (Hematoxylin-eosin staining) images of major organs (heart, liver, spleen, lung, kidneys, brain) were captured.Primarily, the various time points of FL images in vivo were obtained to affirm the accumulation of MRL145-Cy 5.5, whereas, there was almost no accumulation of miR-145-5p lentivirus-Cy 5.5 in the tumor, which suggested MRL145 had excellent tumor targeting ability (Fig. 1e, f ).Collectively, these results demonstrated that MRL145 was expected to exhibit significantly better anti-tumor effect than miR-145-5p-loaded lentivirus and miR-145 over-expressed DNA plasmid.Thus, motivated by these results, we used MRL145 to overexpress miR-145-5p for detailed mechanism studies in the following sections.
The stemness of CSCs is the ability to proliferate indefinitely and CSCs are regarded as the workhorse of tumorinitiating, which can play a key role in initiating tumor formation, recurrence and metastasis [40].Therefore, CSC removal is considered as one of the most important strategies for cancer therapy.Thereafter, we investigated the possible effect of MRL145 on the stemness and proliferation of LCSCs in vitro.LCSCs was transfected with MRL145 and miR-145-5p was over-expressed (Figure S1f ).It was found that compared with the control group, the expression of various proteins involved in regulating proliferation (c-MYC, cyclin D1) was significantly decreased in the MRL145 group (Fig. 2a).Moreover, the results of colony forming assay revealed that the number of colonies of LCSCs was significantly decreased in MRL145 group compared with control group (Fig. 2b).We also observed that overexpression of miR-145-5p significantly promoted the cell proliferation as observed by Cell-Counting-Kit-8 (CCK8) assay in the MRL145 group (Fig. 2c).These results showed that MRL145 can inhibit the proliferation of tumor stem cells in vitro.The self-renewal activity of LCSCs includes both proliferation and stemness.However, the effect of MRL145 on LCSCs stemness was still unknown.To further examine the effects of MRL145 on LCSCs self-renewal activity, we examined the impact of MRL145 modulation on the cell stemness of LCSCs.The protein levels of various stemness associated genes (CD133, CD90, Nanog and Oct4) were downregulated in MRL145 in vitro (Fig. 2d).In addition, The RT-PCR results revealed that miR-145-5p was able to attenuate mRNA levels of CD133, CD90, Nanog and Oct4 (Fig. 2f ).To further assess whether MRL145 can affect LCSCs self-renewal, cell spheroid-formation experiment was performed.MRL145 significantly suppressed the cell sphericity of LCSCs (Fig. 2e).These findings indicated that MRL145 inhibited the stemness of LCSCs and miR-145 can play a vital role as a tumor suppressor which suggested its potential as a novel therapeutic target for HCC.
Another key observation that emerged from our study was that MRL145 reduced EMT in LCSCs.The results of transwell migration and invasion assays indicated that MRL145 decreased the LCSCs invasion as well as migration (Fig. 2g), and it significantly down-regulated the expression of N-cadherin, Vimentin, and Snail as observed by RT-PCR (Fig. 2h).Western blot analysis in vitro was utilized to further confirm the inhibitory effects of MRL145 on the EMT markers (Fig. 2i).
Encouraged by our finding, that MRL145 greatly inhibited the stemness, proliferation and EMT of tumor stem cells in vitro, we further proceeded to investigate whether these results could be translated in vivo.We developed a xenograft model in nude mice using LCSCs to examine the therapeutic efficacy in vivo (Fig. 3a).As expected, miR-145-5p significantly suppressed the tumor volume (Fig. 3b, c) and weight in comparison with the controls (Fig. 3e).Moreover, as indicated by the images of the xenograft tumor, the tumor size in MRL145-treated mice was significantly smaller than those in miR-145-5p-lentivirus and agomir treated mice (Fig. 3d).Furthermore, the expression levels of c-MYC, cyclin D1 and Bcl-2 proteins in MRL145-treated mice were significantly reduced, but the expression level of Bax was significantly increased (Fig. 4a), and the proliferation-ki67 IHC (Fig. 4b) and apoptosis-TUNEL staining (Fig. 4c) also indicated that MRL145 repressed the proliferation of LCSCs and promoted the apoptosis of LCSCs in vivo.Furthermore, the levels of stemness associated proteins (CD133, CD90, Nanog, and Oct4) were more downregulated in the MRL145 group in comparison to both the miR-145-5plentivirus and the agomir group (Fig. 4d).While LNPs may have certain limitations, FDA has approved several LNP-formulated RNA therapeutics [41].In this study, MRL145 could more effectively repress the self-renewal of tumor tissues and promote their apoptosis.
As depicted in the western blot data (Fig. 4e, f ), MRL145 significantly down-regulated the expression of N-cadherin, Vimentin, and Snail proteins.Immunohistochemistry for both E-cadherin and N-cadherin was performed and it was observed that mice treated with MRL145 exhibited lower N-cadherin expression (Fig. 4g, h) but higher E-cadherin expression (Fig. 4g, i), which suggested that MRL145 could effectively prevents EMT in LCSCs under in vivo settings.These results established that MRL145 more effectively repressed cell migration, invasion, all of which may be related to resistance of the cells to EMT process, and self-renewal.These findings revealed that miR-145 could act as an important tumor suppressor and liposomes can effectively deliver miR-145-5p to the tumor sites, thereby resulting in potent antitumor efficacy.
Thereafter, the toxicity of MRL145 towards the body's major organs was examined and depicted in Figure S2.When compared to the control group, histological morphology of different organs and the mice's body weights witnessed no obvious change in Figure S2b, HE staining (Figure S2a).The concentrations of Creatinine (CRE) (Figure S2c), blood urea nitrogen (BUN) (Figure S2d), aspartate aminotransferase (AST) (Figure S2e), and alanine aminotransferase (ALT) (Figure S2f ), showed no significant alterations between the MRL145 group and the control group.These results demonstrated that the MRL145 could form the basis of safe and efficacious therapy for solid tumors, thus suggesting that the application of agomir in vivo might lead to few toxic effects.The study indicated that MRL145 exhibited higher infection efficiency in LCSCs cells, and promising safety was obtained, which can be used as a prospective novel cancer therapy on LCSCs growth in vivo.The prominent advantage of miRNA-liposomes is that it organically integrates the targeting initiative of liposomes, delivery efficiency of cationic liposomes, and high loading efficiency of lentivirus for miRNA.Thus, MRL145 could form the

MiR-145-5p can negatively target COL4A3
It has been established that miRNAs can pair with 3' or 5' UTR untranslated region of its target gene to inhibit the post-transcriptional translation and thereby regulate gene expression.To investigate the mechanism underlying miR-145-5p's effect on LCSCs, we first sequenced MRL145 and control group by mRNA sequencing (Fig. 5a.We noticed that ANO2, COL4A3 and COL4A4 were significantly downregulated in miR-145-5p overexpression LCSCs (Figure S3a-c) (p < 0.05).TIMER2 was used to analyze the expression of COL4A3 in HCC.COL4A3 was found to be significantly upregulated (Figure S3f ) and in the UALCAN database, patients with high expression of COL4A3 showed significantly lower survival rate (Ualcan.path.uab.edu/analysis)(Figure S3g).Besides, immunohistochemical staining of liver cancer patient tumors also indicated significant overexpression of COL4A3 in HCC (Figure S3d, h).Interestingly, the expression level of COL4A3 in LCSCs was significantly higher than that in HCCLM3 cells (Figure S3e, i).
To further explore whether miR-145-5p in LCSCs could be associated with COL4A3, miR-145-5p downstream gene targets were examined, using two different miRNA targeting prediction tools: TargetScan (TargetScan Human 7.2 predicted targeting of human COL4A3).COL4A3 possesses a predicted consequential pairing with miR-145-5p at nucleotide positions 484-490 (Fig. 5b).In addition, a dual-luciferase reporter assay in LCSCs was performed.It was found that compared with control-transfected LCSCs, the luciferase activity of wild-type COL4A3 following transfection with the miR-145-5p mimics was markedly reduced (Fig. 5c).Subsequently, whether miR-145-5p could also regulate COL4A3 expression at the cellular level was investigated.RT-PCR and western blot results demonstrated that the overexpression of miR-145-5p could significantly downregulate COL4A3, both at protein (Fig. 5d, e) (p < 0.05) and mRNA (Fig. 5f ) (p < 0.05) levels in LCSCs.As a result, it was concluded that miR-145-5p could directly target COL4A3 in LCSCs.

MiR-145-5p inhibited the biological behavior of LCSCs by targeting COL4A3
Given that miR-145-5p can hindere the self-renewal of LCSCs, we next analyzed that whether these results could be regulated through COL4A3.In contrary, the effect of miR-145-5p on LCSCs proliferation should be reversed, when LCSCs co-treated with miR-145-5p mimics and PcDNA3.1-COL4A3,similar to that observed in 5-Bromo-2-deoxyUridine (BrdU) (Fig. 6a) and a colony formation assay (Fig. 6c).The rescues assays indicated that miR-145-5p mimics inhibited the expression of c-MYC and cyclin D1 proteins (Fig. 6b).Moreover, we used LCSCs to develop a xenograft mouse model.Micebearing tumors were randomly grouped and treated with COL4A3 shRNA lentivirus 100 μL, respectively thrice every two days.As expected, COL4A3 shRNA significantly suppressed tumor weight compared with the control group (Figure S3k), as indicated by the images obtained from the xenograft tumor (Figure S3j).
To further assess whether miR-145-5p and COL4A3 are required for the maintenance of LCSCs self-renewal, cell spheroidization experiment was performed.It was noted that MiR-145-5p inhibited cell sphericity of LCSCs and COL4A3 reversed the effect of miR-145-5p on the cell sphericity (Fig. 6d).Additionally, results of western blot and RT-PCR experiment showed that miR-145-5p mimics inhibited the stemness of LCSCs.Interestingly, COL4A3 overexpression reversed the effect of miR-145-5p mimics on LCSCs stemness (Fig. 6e, f ) and similar effect was also observed on cell metastasis as well as invasion.Transwell migration and invasion assay (Fig. 6g) results showed that overexpressed COL4A3 reversed the impact of miR-145-5p in preventing metastasis and invasion of LCSCs.Collectively, LCSCs co-treated with PcDNA3.1-COL4A3 and miR-145-5p mimics could down-regulate the expression of the E-cadherin but upregulate the expression of N-cadherin, Vimentin, and Snail at both mRNA (Fig. 6h) and protein levels (Fig. 6i).These findings supported that hypothesis that up-regulation of miR-145-5p interfered with the biological behavior of LCSCs via targeting COL4A3.

MiR-145-5p inhibited the Wnt/β-catenin pathway by targeting COL4A3
A number of prior reports have suggested that attenuation of Wnt/β-catenin pathway can lead to a decrease in cell viability and self-renewal of liver cancer stem cells [42,43].Additionally, Wnt/β-catenin pathway has also been implicated in maintenance of normal stem cell phenotype and tumorigenesis in HCC [44,45].Thus, targeted inhibiting of Wnt/β-catenin pathways in LCSCs could serve as a potential strategy for HCC treatment.So, we examined the effect of miR-145-in modulating Wnt/β-catenin pathway in LCSCs.When the Wnt pathway is triggered, the β-catenin/TCF complex can activate the transcription of Wnt pathway targeted genes, including c-MYC and cyclinD1 [46,47].We examined the expression levels of WNT-associated proteins using western blot analysis in LCSCs and found that the expression level of β-catenin, phosphorylated GSK-3β at ser 9 (p-GSK-3β S9), cyclin D1, c-MYC, and TCF4 proteins were significantly decreased in LCSCs cells transfected with miR-145-5p mimics in comparison to the control group in vitro (Fig. 7a) and in vivo (Fig. 7b), thus indicating that miR-145-5p repressed the activation of the Wnt/β-catenin pathway and then hindered the proliferation of LCSCs.Thereafter we transfected LCSCs with COL4A3 pcDNA 3.1 vector.The results of LCSCs transfected with PcDNA3.1-COL4A3 were observed to be opposite compared with miR-145-5p mimics group (Fig. 7a).The accumulation of β-catenin in the nucleus is indicative of activation of canonical Wnt pathway [48].Mechanistically, GSK3 phosphorylation can stabilize the β-catenin [49], which can then translocate into the nucleus and complexes with TCF to activate canonical Wnt pathway [50].Here, we found that COL4A3 can significantly up-regulate the nuclear accumulation of β-catenin thereby resulting in the activation of Wnt/βcatenin pathway.As depicted in Fig. 7c, higher levels of nuclear β-catenin were detected in LCSCs cells transfected with PcDNA3.1-COL4A3.Together, these results suggested that COL4A3 can bind directly to p-GSK-3β S9 or another member of the complex, thus disabling this critical β-catenin degradation enzyme, and leading to increased accumulation, nuclear translocation, and transcriptional activity of β-catenin to activate the Wnt/βcatenin pathway.Moreover, miR-145-5p directly targeted COL4A3 to down-regulate the nuclear accumulation of β-catenin thereby resulting in the inactivation of the Wnt/β-catenin pathway.
Interestingly, a previous study has shown that the Wnt/β-catenin pathway plays a crucial role in maintaining stemness in liver cancer [42], so whether this pathway was involved in COL4A3-mediated self-renewal activity, tumorigenesis was further investigated.The Wnt pathway inhibitor (ICG-001) was used in COL4A3 overexpressed LCSCs.It was found that COL4A3 overexpression exerted the ability to increase the protein level of β-catenin, cyclin D1, c-MYC, and TCF4 whereas, ICG depressed the protein level of β-catenin, cyclin D1 and TCF4 in COL4A3 overexpressed LCSCs (Fig. 7d).More intriguingly, COL4A3 treatment rescued the stemness inhibition caused by ICG by accelerating the protein expression (Fig. 7e) and mRNA level of Oct4, CD90, CD133 in comparison to ICG group (Fig. 7f ).Moreover, COL4A3 significantly promoted the invasion and migration of LCSCs compared with the ICG group (Fig. 7g).In terms of mechanism, COL4A3 elevated the mRNA levels of E-cadherin (Fig. 7h) N-cadherin (Fig. 7i) and the protein expression of N-cadherin, Vimentin, and Snail (Fig. 7j) in comparison with ICG group.Additionally, COL4A3 overexpression rescued the metastasis inhibition of ICG., Overall, these results suggested that COL4A3 affected the self-renewal and metastasis of LCSCs by targeting activation of Wnt/β-catenin pathway.The data suggested that miR-145-5p inhibited activation of Wnt/β-catenin pathway by suppressing COL4A3 expression and then hindered the proliferation, selfrenewal, and EMT ability of LCSCs.

MiR-145-5p inhibited autophagy by targeting COL4A3-Gli3-VMP1
GSK-3β signaling pathway has been reported to exert dual regulatory function on the process of autophagy [51], which is considered as a major cause of survival and chemotherapy resistance in CSCs [52].Moreover, a recent study has indicated that autophagy can promote the endothelial cells differentiating from CSCs, and CSCs activation is largely dependent on autophagy [53].It has been also reported that autophagy can lead to a substantial increase in cell self-renewal capacity of LCSCs.[54].Thus, after LCSCs were treated with autophagy activator rapamycin (Rapa) or autophagy inactivator (3-MA), the levels of the cell proliferation-related genes (cyclin D1 and c-MYC) proteins were more intensively down-regulated in 3-MA group in comparison to that in the Rapa group (Figure S4a).The colony formation results indicated that the number of colonies of LCSCs significantly decreased in 3-MA group, but, the number of colonies of LCSCs significantly increased in Rapa group upon comparison with the control group (Figure S4b, f ).More intriguingly, Rapa significantly promoted the expression of the various stemness markers (Figure S5c-d).In addition, Rapa promoted the cell sphericity of LCSCs but 3-MA suppressed the cell sphericity (Figure S4e).These results indicated that the activation of autophagy could effectively protect the self-renewal capacity of LCSCs.
To investigate how COL4A3 can potentially regulate autophagy, we envisaged a possible relationship between GSK-3β and autophagy, because COL4A3 could repress the activity of GSK-3β through promoting the expression of p-GSK-3β.It has been reported GSK-3β can cause phosphorylation of Gli3, which can then suppress the activation of Hh pathway [57], thus repressing autophagy of cancer cells through inhibiting the expression of VMP1 [58,59].Thus, we speculated that miR-145-5p can mediate suppression of COL4A3 regulated autophagy in LCSCs by targeting Gli3/VMP1 axis.We found in this study that compared with the control group, p-GSK-3β S9 overexpression promoted the expression of Gli3, while transfection of Gli3 siRNA repressed the expression of Gli3 to activate the Hh pathway (Fig. 8e).More intriguingly, p-GSK-3β S9 overexpression reversed the inhibitory effect of Gli3 siRNA on the Hh pathway (Fig. 8e), which indicated that p-GSK-3β could activate the Hh pathway.Loss of Vacuole membrane protein 1 (VMP1, the endoplasmic reticulum (ER)-localized autophagy protein) can lead to the autophagosome formation [59].Interestingly, in this study, p-GSK-3β S9 overexpression also promoted the expression of VMP1, but transfection with Gli3 siRNA repressed the expression of VMP1.Moreover, GSK-3β ser9 overexpression reversed the inhibition of Gli3 siRNA on VMP1 (Fig. 8e).In addition, autophagic capacity of p-GSK-3β overexpressed LCSCs was significantly increased, whereas it was reduced in LCSCs treated with Gli3 siRNA.Western blot (Fig. 8e) results indicated that Gli3 siRNA reduced the protein level of LC3II, but enhanced the protein level of p62.Besides, there were more GFP-LC3 signal observed in p-GSK-3β overexpressed LCSCs in comparison with Gli3 siRNA group (Fig. 8f ).Above all, these results suggested that COL4A3 could promote the autophagy by targeting p-GSK-3β through Gli3/VMP1.These findings are consistent with the aforementioned studies on the role of autophagy in cancer stem cell progression [60][61][62] (Figs. 7 and 8) and implies a possible association between autophagy induced by COL4A3 in LCSCs.

Conclusion
In conclusion, miR-145-5p lentivirus-liposome was found to effectively deliver miR-145-5p into LCSCs cells and the tumor site.MiR-145-5p could inhibit LCSCs properties through modulating the Wnt/β-catenin and autophagy pathway.Importantly, miR-145-5p could specifically bind to COL4A3, and then inhibit the activity of GSK3β to suppress the Wnt/β-catenin pathway.Moreover, COL4A3 could regulate VMP1-Gli3-mediated autophagy.The discovery of miR-145-5p/COL4A3/ Wnt/β-catenin and autophagy regulatory network could be potentially used as novel therapeutic targets for effective liver cancer treatment.Future investigations utilizing LCSCs overexpressing miR-145-5p for the treatment of HCC and/or COL4A3 preclinical models could be beneficial to verify our current findings.Our observations have shed light on a novel mechanism of liver CSCs and might be potential therapeutic targets against liver cancer.In addition, it is envisioned that further development of gene-editing therapies together with efficient delivery nanotechnologies would contribute to expedite clinic translation and even improve prognosis of patients bearing cancers in future.

Fig. 1
Fig. 1 Characterization of MRL145.a TEM images of MRL145.b HEK-293T cells were transduced with miR-145-5p lentivirus and MRL145, scale bar = 50 µm.c The significant signal intensity of d. d FL images in vivo were obtained at various time points to affirm the accumulation of MRL145.e The FL images of major organs (heart, liver, spleen, lung, kidneys, tumor).f The significant signal intensity of the tumor

Fig. 2
Fig. 2 MRL145 inhibited the stemness and biological behaviour of LCSCs.a Western blot analysis of c-MYC and cyclin D1 proteins in LCSCs cells.b The colony formation assays in LCSCs cells.c Cell growth was measured by CCK8 assay.d Western blot analysis of CD133, CD90, Nanog, Oct4 in LCSCs cells.e The formation process of the tumor stem cell microspheres, scale bar = 100 µm.f The mRNA expression of stemness genes (CD133, CD90, Nanog and Oct4) in LCSCs cells by RT-PCR.g Transwell assay was used to measure the migration and invasion ability of LCSCs cells.h RT-PCR analysis of N-cadherin, E-cadherin, Vimentin and Snail in LCSCs cells.i Western blot analysis of N-cadherin, E-cadherin, Vimentin and Snail proteins in mice xenografted tumor.All the data was presented as mean ± SD. (n = 3).*P < 0.05, ***P < 0.001

Fig. 3
Fig. 3 MRL145 inhibited the growth of LCSCs cells in a xenograft tumor model.MRL145 suppressed the growth of LCSCs cells in a xenograft tumor model.a Mice bearing tumors were randomly grouped and treated with miR-145-5p-5p lentivirus 100 μL, MRL145 100 μL, 80 mg/kg body weight miR-145-5p agomir, respectively every two days for five times.b The photos of mice.c The tumor volume measurement was performed every other day.d The representative photographs of isolated tumors at day 15 after treatment.e Tumor weight of mice at day 15 after treatment.All the data has been presented as mean ± SD. (n = 3).**P < 0.01, ***P < 0.001

Fig. 4
Fig. 4 MRL145 inhibited the stemness and biological behaviour of LCSCs in vivo.a Western blot analysis of c-MYC, cyclin D1, Bcl2 and Bax proteins in mice xenografted tumor.b Protein expression of ki67 as measured by IHC (Scale bar = 100 µm).c The images of TUNEL assay obtained from the different groups after the treatment (scale bar = 40 µm).d Western blot analysis of proteins associated with tumor stemness CD133, CD90, Nanog, Oct4 in the tumor tissues derived from mouse.e Western blot analysis of N-cadherin, E-cadherin, Vimentin, Snail proteins in the tumor tissues.f Quantification of e. g Expression of N-cadherin and E-cadherin proteins as measured by IHC (Scale bar = 100 µm).h, i Statistical analysis of g.All the data has been presented as mean ± SD. (n = 3).*P < 0.05, ***P < 0.001

Fig. 5
Fig. 5 COL4A3 could be a direct miR-145-5p-5p target.a The mRNA sequence in LCSCs.b Luciferase reporter contained the miR-145-5p-5p targeting sequences and mutated version of the COL4A3 3′ UTR wild type, including the site where the binding agent was altered (red).The luciferase reporter gene containing wild-type (WT) and mutant-type (Mut) COL4A3 3′ UTR was co-transfected into HEK-293 T cells by simulating NC or miR-145-5p-5p mimics.c Luciferase activity was measured and normalized 72 h after transfection.d, e The protein level of COL4A3 protein level of LCSCs was detected by western blotting.f COL4A3 mRNA levels in the different groups were analyzed by RT-PCR.All the data has been presented as mean ± SD. (n = 3).*P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6
Fig. 6 MiR-145-5p-5p inhibited the stemness and biological behaviour of LCSCs by targeting COL4A3.a Cell growth was measured by BrdU staining, scale bar = 100 µm.b Western blot analysis of c-MYC, cyclin D1 proteins in LCSCs cells treated with miR-145-5p mimics or co-treated with miR-145-5p mimics and PcDNA3.1 COL4A3.c The colony formation assays in LCSCs cells.d The process of tumor stem cell microsphere formation, scale bar = 100 µm.Images correspond to day 7. e Western blot analysis of CD133, CD90, Nanog and Oct4 in LCSCs cells.f The mRNA expression of stemness genes (CD133, CD90, Nanog and Oct4) in LCSCs cells as measured by RT-PCR.g Transwell assay was used to detect the effect of miR-145-5p and COL4A3 on migration and invasion ability of LCSCs cells.h The mRNA expression of N-cadherin, E-cadherin, Vimentin and Snail in LCSCs cells as analyzed by RT-PCR.i Western blot analysis of N-cadherin, E-cadherin, Vimentin and Snail in LCSCs cells.All data has been presented as mean ± SD. (n = 3).*P < 0.05, ***P < 0.001